Objectives: The aim of this study was to assess the usefulness of high-resolution melting (HRM) curve analysis on the LightCycler 480 PCR system as a tool for the correct identification of Bacillus anthracis and to assess its utility as a method to recognise the Ames strain, the strain used in the 2001 anthrax attacks in the USA.

Methods: DNA from nine bacterial isolates [eight B. anthracis and one B. cereus DSM 4312], including one strain originating from an American diplomatic postal bag in December 2001 in Vienna [1], was isolated using the Qiagen Blood & Body Fluid Spin Protocol as described by the manufacturer. All B. anthracis isolates were subtyped by VNTR analysis as described previously using a 25 VNTR system [2]. For HRM curve analysis six specific primer combinations were chosen and tested for the identification of the Ames strain by specific single nucleotide polymorphisms [3] using the LightCycler 480 High Resolution Melting Master as described by the manufacturer. The obtained data were analysed with the HRM gene scanning software (Roche).

Results: VNTR analysis confirmed the 2001 'postal bag' isolate as a B. anthracis Ames strain and the other B. anthracis isolates as 'non-Ames' genotypes. HRM curve analysis allowed 1) the differentiation of B. cereus DSM 4312 from the B. anthracis isolates and 2) the rapid and accurate discrimination of the B. anthracis Ames strain from the other seven B. anthracis isolates. Of interest, the VNTR data from three B. anthracis isolates from Tyrol, Austria were shown to be European B2 strains and extremely similar to French and Italian B2 genotypes. Two B. anthracis isolates from the country of Georgia were also subtyped; one was identical to the vaccine strain 34F2 Sterne, and the other (411-G) was an A3a isolate.

Conclusion: We were able to show that HRM curve analysis is a fast, simple and cost effective screening method for identification of B. anthracis strains. HRM curve analysis, coupled with strain-specific SNP signatures, allows differentiation of the Ames strain from other B. anthracis strains using a single PCR. As the source of the intentional release of B. anthracis spores in 2001 has never been elucidated, the ability to identify or exclude the presence of the Ames strain in clinical and environmental samples is of great importance.

References

1. Allerberger F, et al. (2002) Lancet 359: 710

2. Lista F, et al. (2006) BMC Microbiol. 6: 33

3. Van Ert MN, et al. (2007) J. Clin Microbiol. 45: 4753

Session Details

Date:

19/04/2008

Time:

00:00-00:00

Session name:

18th European Congress of Clinical Microbiology and Infectious Diseases